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Stable reference genes for the measurement of transcript abundance during larval caste development in the honeybee

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Abstract

Many genes are differentially regulated by caste development in the honeybee. Identifying and understanding these differences is key to discovering the mechanisms underlying this process. To identify these gene expression differences requires robust methods to measure transcript abundance. RT-qPCR is currently the gold standard to measure gene expression, but requires stable reference genes to compare gene expression changes. Such reference genes have not been established for honeybee caste development. Here, we identify and test potential reference genes that have stable expression throughout larval development between the two female castes. In this study, 15 candidate reference genes were examined to identify the most stable reference genes. Three algorithms (GeNorm, Bestkeeper and NormFinder) were used to rank the candidate reference genes based on their stability between the castes throughout larval development. Of these genes Ndufa8 (the orthologue of a component of complex one of the mitochondrial electron transport chain) and Pros54 (orthologous to a component of the 26S proteasome) were identified as being the most stable. When these two genes were used to normalise expression of two target genes (previously found to be differentially expressed between queen and worker larvae by microarray analysis) they were able to more accurately detect differential expression than two previously used reference genes (awd and RpL12). The identification of these novel reference genes will be of benefit to future studies of caste development in the honeybee.

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Acknowledgments

RCC would like to thank Caroline Walker for advice and discussion that led to the development of this study. All authors would like to thank Frans Laas and Bettabees for help obtaining larval samples. This work was funded by the Royal Society of New Zealand Marsden Fund Grant (UOO0707) to P.K.D.

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Authors and Affiliations

  1. Laboratory for Evolution and Development, National Research Centre for Growth and Development and Genetics Otago, Dunedin, New Zealand

    Rosannah C. Cameron, Elizabeth J. Duncan & Peter K. Dearden

  2. Biochemistry Department, University of Otago, Dunedin, New Zealand

    Rosannah C. Cameron, Elizabeth J. Duncan & Peter K. Dearden

Authors
  1. Rosannah C. Cameron
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  2. Elizabeth J. Duncan
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  3. Peter K. Dearden
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Correspondence to Peter K. Dearden.

Additional information

Gènes de référence stables pour mesurer l’abondance de transcrits chez les larves d’abeilles durant le développement des castes

Développement/ caste/ développement larvaire/ expression génique/ PCR quantitative

Stabile Referenzgene für quantitative Transkriptbestimmungen in der Kastenentwicklung von Honigbienenlarven

quantitative RT-PCR/ Referenzgene/ Kastenentwicklung

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Supplementary Figure 1

Expression of the 15 candidate genes in queens and workers throughout larval development. Raw Cq values are represented in box and whisker plots for queens and workers. The y-axis indicates the Cq values and the x-axis indicates the caste and time point sampled (Q = queens, W = workers). A two-way ANOVA and LSD post-hoc test were used to determine time points where queens and workers showed a significant difference in gene expression. P values < 0.05 are indicated by a single asterisk and P values < 0.01 by two asterisks. (DOC 19 kb)

Supplementary Figure 1b

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ESM 1

(DOCX 77 kb)

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Cameron, R.C., Duncan, E.J. & Dearden, P.K. Stable reference genes for the measurement of transcript abundance during larval caste development in the honeybee. Apidologie 44, 357–366 (2013). https://doi.org/10.1007/s13592-012-0187-0

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